Curcumin coated magnetite nanoparticles for biomedical applications

ABSTRACT

The present invention discloses biocompatible, stable curcumin or its derivatives coated ultra-small super paramagnetic iron oxide nanoparticles (USPION) for biomedical applications. Disclosed herein is also a simple one-pot process for the synthesis of biocompatible, stable curcumin or its derivatives coated ultra-small superparamagnetic iron oxide nanoparticles in absence of a linker or binder. The curcumin or its derivatives coated ultra-small super paramagnetic iron oxide nanoparticles of the present invention retains the medicinal, radical scavenging and fluorescence properties of curcumin.

This application is a 35 U.S.C. 371 national stage filing of PCTApplication No. PCT/IN2013/000030, entitled “Curcumin Coated MagnetiteNanoparticles for Biomedical Applications” filed on Jan. 17, 2013, whichclaims the benefit of Indian Application No. 138/DEL/2012 filed on Jan.17, 2012, both of which are incorporated herein in their entirety byreference.

TECHNICAL FIELD OF INVENTION

Present invention relates to curcumin coated magnetite nanoparticles,which are biocompatible, stable curcumin or its derivatives coatedultra-small superparamagnetic iron oxide nanoparticles (USPION) forbiomedical applications. The invention further relates to a simpleone-pot process for the synthesis of biocompatible, stable curcumin orits derivatives coated ultra-small superparamagnetic iron oxidenanoparticles in absence of a linker or binder. The curcumin or itsderivatives coated ultra-small super paramagnetic iron oxidenanoparticles of the present invention retains the medicinal, radicalscavenging and fluorescence properties of curcumin.

BACKGROUND OF THE INVENTION

Metal oxide nanoparticles are one of the most significant contributorstowards the revolutionary change of nanostructured science andtechnology. Out of many metal oxides, multifunctional iron oxides havepaved the way towards applications such as ferrofluids, colour imaging,recording media and magnetic refrigeration. Thrust areas of applicationsof iron oxides in biological field include contrast agents in MRI, inhyperthermia treatment and drug delivery. The prerequisite for all theseapplications are smaller size, superparamagnetism, biodegradability,monodispersed, water dispersible particles which have higher stabilityat room temperature (±20 degrees) and at physiological pH. The smallersize allows nanoparticles to cross through the cellular membranes andavoid the detection by the reticuloendothelial system as well as allowlonger blood residence time and subject to rapid renal elimination.

Uncoated magnetite nanoparticles do not form stable dispersions in waterat physiological pH. Apart from high surface energy (which is due tohigh surface area to volume ratio), magnetic nanoparticles also havedipole-dipole interaction which tends the particles to aggregatecompared to other non-magnetic counterparts. Thus the particles have tobe stabilized which can be done in two ways: either by stericstabilization or by electrostatic stabilization. Steric stabilization isattained by attaching long chain surfactants on to the surface ofnanoparticles to prevent the particles from approaching closer, therebyoverwhelming the attractive components which tend the particles toaggregate. Electrostatic stabilization is achieved by introducingsurface charge onto the nanoparticles, which results in electrostaticrepulsion of nanoparticles, hence leading to stabilization of thedispersion.

Number of synthetic strategies are available for the synthesis ofparticles in organic media at elevated temperatures which leads tohydrophobic fairly monodispersed nanoparticles. Palma et al in ChemMater 2007, 19, 1821-1831 reported synthesis where hydrophobic ligandsare exchanged with hydrophilic to make these particles waterdispersible, but the synthesis steps include high temperature as well asharmful organic solvents. Another problem with high temperaturesynthesis is to keep the bio-molecules at the surface of the particlesbiologically active at the synthesis temperature or on cooling at roomtemperatures. Other methods include microemulsion, hydrothermal orsonochemical, which are also not effective in producing the particleswith requisite properties.

In biomedical applications, another prerequisite of the nanoparticles islarge surface area to volume ratio that allows for the increased loadingof therapeutics thereby making them useful in drug delivery.

Curcumin (CUR) a yellow polyphenol compound found in the rhizomes of theplant curcuma longa is known for its excellent anti-oxidant,anti-cancer, anti-inflammatory, and anti-microbial activities, whichmakes it a promising candidate for coating on iron oxide nanoparticlesfor biomedical applications. The coating of curcumin on iron oxidenanoparticles with linkers like oleic acid, chitosan and silica has beenreported.

Tran et at in Colloids Surf. A: Physicochem. Eng. Aspects, 2010, 371,104-112 has reported the synthesis of Fe₃O₄ nanoparticles-curcuminconjugate where curcumin is attached to nanoparticles by using linkerslike chitosan and oleic acid. In the above mentioned work, curcumin isindirectly attached onto the magnetite nanoparticles via a linker. Alsothe author mentions that curcumin is only being adsorbed onto thechitosan or oleic acid coated magnetite nanoparticles. The linkermentioned could not improve the water dispersibility of the preparedfluid.

An article titled “Curcumin-loaded magnetic nanoparticles for breastcancer therapeutics and imaging applications” by Murali M Yallapu, ShadiF Othman et. al. in Int J Nanomedicine. 2012; 7: 1761-1779 having doi:10.2147/IJN.S29290 disclose formulation composed of an iron oxide corecoated with β-cyclodextrin (CD) and pluronic F68 polymer (polyethyleneoxide-co-polypropylene oxide-co-polyethylene oxide) and loadinganticancer drug curcumin. The article further discloses preparation ofmagnetic nanoparticles comprising dissolving cyclodextrin, solution ofiron(3+) and iron(2+) ions (molar ratio 2:1) in water, ammoniumhydroxide, pluronic polymer F68 stirring, washing drying to obtainmagnetic nanoparticles (MNC) and followed by loading of curcuminsolution in acetone, facilitating the penetration of curcumin molecules(CUR) into the CD or CD-F68 polymer layers in the formulation. MNP-CURexhibited individual particle grain size of ˜9 nm and hydrodynamicaverage aggregative particle size of ˜123 nm.

Article titled “Superparamagnetic iron oxide nanoparticles: magneticnanoplatforms as drug carriers” by Wahajuddin and Sumit Arora et. al inInt J Nanomedicine. 2012; 7: 3445-3471 having doi: 10.2147/IJN.S30320relates to superparamagnetic iron oxide nanoparticles (SPIONs) which aresmall synthetic γ-Fe2O3 (maghemite) or Fe3O4 (magnetite) particles witha core ranging between 10 nm and 100 nm in diameter as novel drugdelivery vehicles. The magnetite nanoparticles are obtained byco-precipitation of iron(3+) and iron(2+) ions (molar ratio 2:1) whichare further coated with suitable polymers, liposomes, dendrimers etc.Drug loading is achieved either by conjugating the therapeutic moleculeson the surface of SPIONs or by coencapsulating drug molecules along withmagnetic particles within the coating material envelope.

Article titled “Biomedical properties and preparation of ironoxide-dextran nanostructures by MAPLE technique” by Carmen S Ciobanu,Simona L Iconaru et. al in Chemistry Central Journal 2012, 6:17doi:10.1186/1752-153X-6-17 relate to dextran coated iron oxidenanoparticles thin films. The dextran-iron oxide continuous thin filmsare obtained by MAPLE technique from composite targets containing 10 wt.% dextran as well as 1 and 5 wt. % iron oxide nanoparticles synthesizedby co-precipitation method. The particle sized calculated was estimatedat around 7.7 nm.

Article titled “Encapsulation and Sustained Release of Curcumin usingSuperparamagnetic Silica Reservoirs” by Suk Fun Chin, K. SwaminathanIyer et. al having DOI: 10.1002/chem.200802747 disclose synthesis ofFe3O4 nanoparticles by using spinning-disc processing with Fe²⁺/Fe³⁺ inaqueous NH₄OH. The as-synthesized Fe₃O₄ nanoparticles were 8-10 nm insize. The article further discloses encapsulation of Fe₃O₄ nanoparticlesand curcumin in mesoporous silica capsules.

The prior art reports on the synthesis of conjugates of magneticnanoparticles and curcumin with a linker or binder are observed toneither increase the stability of the resulting magnetic fluid nor dothey enhance the property of the magnetic iron oxide core ornon-magnetic curcumin shell.

OBJECTS OF THE INVENTION

Main object of the present invention is to provide biocompatiblecurcumin or its derivatives directly coated on magnetite nanoparticlesfor biomedical applications.

Another object of the present invention is to provide a simple one-potprocess for direct coating of the curcumin onto the surface of thenanoparticle so that the hydrodynamic size can be minimized which isdesirable for biomedical applications.

Yet another object of the present invention is to provide a process forthe synthesis of biocompatible ultra-small iron oxide nanoparticles atlow temperature thereby reducing the possibility for decomposition ofthe biomolecule used for coating.

SUMMARY OF THE INVENTION

Accordingly, present invention provides a curcumin coated magnetitenanoparticles, which are biocompatible, stable curcumin or itsderivatives coated ultra-small super paramagnetic iron oxidenanoparticles devoid of any linker or binder that retains the medicinal,radical scavenging and fluorescence properties of curcumin, forbiomedical applications.

In an embodiment of the present invention, curcumin is directly coatedon ultra-small super paramagnetic iron oxide nanoparticles.

In one embodiment of the present invention, curcumin coated ultra-smallsuper paramagnetic iron oxide nanoparticles is of 3 nm size.

In an embodiment, present invention provides a simple one-pot processfor the synthesis of curcumin coated magnetite nanoparticles, which arebiocompatible, stable curcumin or its derivatives coated ultra-smallsuperparamagnetic iron oxide nanoparticles in absence of a linker orbinder comprising;

-   -   a. dissolving aqueous mixture of FeCl₃.6H₂O and FeCl₂.4H₂O in        the molar ratio ranging between 1.5:1 to 2:1 in a base under        inert atmosphere and stirring until complete formation and        growth of magnetite nanoparticles;    -   b. adding dilute mineral acid to the solution as obtained in        step (a) until pH 9;    -   c. adding drop wise curcumin solution dissolved in a base to the        solution of step (b) under inert atmosphere, maintaining the pH        9 followed by stirring the dispersion at a temperature in the        range of 50-100° C. followed by cooling;    -   d. dialysing the dispersion of step (c) against water to remove        excess curcumin and drying in vacuum to obtain the product.

In another embodiment of the present invention, the base used isselected from alkali hydroxide preferably ammonium hydroxide.

In yet another embodiment of the present invention, the mineral acidused is preferably nitric acid.

In yet another embodiment, present invention provides a method formagnetic as well as fluorescent imaging and other biomedicalapplications comprising providing curcumin coated magnetitenanoparticles, which are biocompatible, stable curcumin or itsderivatives coated ultra-small iron oxide nanoparticles devoid of anylinker or binder, the curcumin coated magnetite nanoparticles being usedas a contrast agent.

In yet another embodiment, present invention provides a method fordelivering curcumin to a subject in need thereof comprisingadministering curcumin coated magnetite nanoparticles, which arebiocompatible, stable, curcumin or its derivatives coated ultra-smalliron oxide nanoparticles devoid of any linker or binder.

In yet another embodiment, present invention provides use of curcumincoated magnetite nanoparticles, which are biocompatible, stable curcuminor its derivatives coated ultra-small iron oxide nanoparticles devoid ofany linker or binder as contrast agent for magnetic as well asfluorescent imaging and other biomedical applications.

In yet another embodiment, present invention provides use of curcumincoated magnetite nanoparticles, which are biocompatible, stable,curcumin or its derivatives coated ultra-small iron oxide nanoparticlesdevoid of any linker or binder for delivering curcumin to a subject inneed thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts X-ray diffraction (XRD) patterns of standard JointCommittee on Powder Diffraction Standards (JCPDS) data (19-0629) ofFe₃O₄ (a), M0, M1 and M2 samples.

FIG. 2 depicts Transmission electron microscopy (TEM) image of curcumincoated ultra-small super paramagnetic iron oxide nanoparticles.

FIG. 3 depicts the Fourier transform infrared spectroscopy (FTIR)spectra of Curcumin (c), M2, M1 and M0 samples.

FIG. 4 depicts Thermo gravimetric analysis (TGA) curve for curcumin (Cor CUM), M2, M1 and M0 samples.

FIG. 5a depicts room temperature Magnetic measurements.

FIG. 5b depicts Zero field cooled (ZFC)/field cooled (FC) curves foruncoated and curcumin coated iron oxide nanoparticles. The measurementswere done in a constant small applied field of 50 Oe.

FIG. 6 (A) UV-Vis spectra of CuF, UnF and Curcumin (CUR);

FIG. 6 (B) Fluorescence absorption spectra of CuF and CUR measured at anexcitation wavelength of 430 nm.

FIGS. 7A and 7B depicts plots to determine R1 and R2 from T1 and T2measured at different concentrations.

FIG. 8 depicts the photographs of the dispersion of curcumin coatednanoparticles and curcumin in DMSO before and after illuminated with aUV lamp at 365 nm, indicating the fluorescence nature of the coatednanoparticles.

DETAILED DESCRIPTION OF INVENTION

The term ‘magnetite nanoparticles’ as used herein mean and refer tosuper paramagnetic iron oxide nanoparticles.

Abbreviations:

M₀—uncoated magnetite sample.

M2 or insitu coated sample—insitu coated superparamagnetic iron oxidenanoparticles.

M1 or CuF—curcumin coated ultra-small superparamagnetic iron oxidenanoparticles (Post synthesis functionalised sample).

Curcumin being non-toxic, biocompatible, fluorescent and having novelmedicinal properties, can act as a multifunctional probe in coatednanoparticles. The uptake of Fe₃O₄-curcumin conjugate can be monitoredby both fluorescent as well as magnetic imaging and hence has advantagesover other coated nanoparticles in biomedical applications. Further,considering the high affinity of metal ions to the hydroxyl groups ofcurcumin, the invention provides a simple process for preparingultra-small superparamagnetic nanoparticles of iron oxide on whichcurcumin or its derivatives can be coated directly without losing themedicinal properties of curcumin.

In accordance with the above, the present invention disclosebiocompatible, stable curcumin or its derivatives coated ultra-smallsuper paramagnetic iron oxide nanoparticles devoid of any linker orbinder, that retains its medicinal, radical scavenging and fluorescenceproperties of curcumin.

The curcumin molecule is directly attached on to the surface ofmagnetite nanoparticles through its enolic hydroxyl group without losingits medicinal properties.

The invention relates to a simple one-pot process for the preparation ofcurcumin or its derivatives coated ultra-small superparamagnetic ironoxide nanoparticles in absence of a linker or binder.

The process employed is a co-precipitation technique since the methoddoes not require any harmful precursors and can be carried out atvarious temperatures to control size, morphology, dispersity etc. Thereaction can be tuned by varying pH of the reaction medium, due to thedifference in acidity of the phenolic and enolic hydroxyl groups. Theprocess allows for direct coating of curcumin on to the surface ofultra-small magnetite nanoparticles while retaining the medicinalproperties of curcumin.

The process includes the following steps:

-   -   1. dissolving aqueous mixture of FeCl₃.6H₂O and FeCl₂.4H₂O in        the molar ratio 2:1 in a base under inert atmosphere and        stirring until complete formation and growth of magnetite        nanoparticles;    -   2. adding dilute mineral acid to the above solution until pH 9;    -   3. adding drop wise curcumin solution dissolved in a base to        solution of step 2, under inert atmosphere, maintaining the pH        9, and stirring the dispersion at a temperature of 50-100° C.        followed by cooling;    -   4. dialysing the dispersion of step 3 against water to remove        excess curcumin and drying in vacuum to obtain the desired        product.

The process is carried out in inert atmosphere, preferably nitrogen orargon atmosphere, to prevent oxidation of magnetite nanoparticles

The base is selected from alkali hydroxides, preferably ammoniumhydroxide and the mineral acid is preferably dil. nitric acid.

Curcumin coated ultra-small superparamagnetic iron oxide nanoparticles(USPIONs) obtained is 3 nm in size.

Accordingly, to a mixture of FeCl₃.6H₂O and FeCl₂.4H₂O (as precursors)in water in a molar ratio 2:1 is added a base under argon atmosphere.The mixture is stirred for complete formation and growth of magnetitenanoparticles. pH of the dispersion is brought to pH 9 by addition ofdilute nitric acid, curcumin dissolved in base is added dropwise to thedispersion while maintaining the pH 9. The temperature of the solutionis slowly raised to a temperature in the range of 50-100° C. and themixture is stirred under argon atmosphere for about 30 min. Theresulting stable dispersion is then cooled to room temperature (21 to40° C.) and dialysed against water in a cellulose membrane to removeexcess curcumin followed by drying in vacuum to obtain the desiredproduct.

The present invention provides the synthesis of uncoated iron oxidenanoparticles by the co-precipitation process described above forcomparison of the XRD pattern of the as synthesized nanoparticles isshown in FIG. 1.

Accordingly, a mixture of FeCl₃.6H₂O and FeCl₂.4H₂O (as iron precursors)in water in the molar ratio 2:1 in a base under nitrogen atmosphere andstirring until complete formation and growth of magnetite nanoparticlesfollowed by adding dilute mineral acid to the above solution until pH 9.

The uncoated and curcumin coated iron oxide nano-particles aredesignated as UnF (M0) and CuF (M1) respectively for further studies.

The uncoated and curcumin coated iron oxide nano-particles arecharacterized using powder X-ray diffraction (XRD) (FIG. 1). The averagecrystallite sizes of uncoated and coated samples are in the range of 7nm and 4 nm respectively. (Particle size decreases for coated particlesbecause coating prevents particle aggregation).

Room temperature magnetic measurements of curcumin coated (CuF) anduncoated (UnF) samples reveal that magnetization of both coated anduncoated samples do not get unsaturated even at a magnetic field of 3 T.Further, no hysteresis loops are observed for both the samples (zerocoercivity) indicating both are super paramagnetic. The magnetization at3 T for uncoated sample is observed to be 30 emu/g and for coated sampleis 11 emu/g. The decrease in saturation magnetization compared to theuncoated sample can be ascribed to the reduced size after coating whichincreases the contribution from the magnetically dead layer from thesurface of the particles and the non-magnetic coating layer (curcumin)over each particle. (The non magnetic dead layer is curcumin whichreduces the overall mass of iron oxide nanoparticles)

The curcumin coated nanoparticles as well as uncoated particles show nocoercivity and remanence, as inferred from M-H measurements at roomtemperature and therefore, are superparamagnetic.

The coating of curcumin onto the magnetite nanoparticles is furtheranalysed by IR spectroscopy which shows significant change in theposition of various peaks in comparison to curcumin as such.

The scavenging property of the as synthesized curcumin coated iron oxidenanoparticles is analysed. The sample is treated with hydrogen peroxideto check whether the radical scavenging property of curcumin isretained. The percentage of H₂O₂ scavenging by curcumin and otherscavengers is calculated using the formulaH₂O₂ scavenging effect (%)=(1−A _(s) /A _(c))×100where A_(c) is the absorbance of the control and A_(s) is the absorbancein the presence of curcumin.

In a particular scavenging activity assay, hydrogen peroxide inphosphate buffer is added to the dispersion of curcumin coated ironoxide nanoparticle (CuF) in phosphate buffer. The mixture is incubatedfor about 15 min and the absorbance of the solution is measured at 230nm of H₂O₂) using a UV-Visible spectrophotometer.

The H₂O₂ scavenging effect of curcumin coated iron oxide nanoparticle(CuF) of the present invention is about 62% as compared to the 81%scavenging activity of 30 ppm curcumin solution confirming the radicalscavenging property of curcumin even after coating on SPIONs.

Since the phenolic group is known for the antioxidant activity ofcurcumin, the study confirms that phenolic hydroxyl group remains freeon the curcumin coated on nanoparticles.

That the scavenging activity of curcumin coated nanoparticle is retainedis further confirmed by XRD, IR and magnetic measurement of H₂O₂ treatedand untreated samples as detailed in the example below.

From the measurements described below, it can be concluded that thecurcumin coated nanoparticles are highly stable and can be reused forperoxide scavenging as well as for other applications as there is nostructural damage observed after peroxide treatment.

The thermogravimetric studies of the synthesized samples are carried outbetween 100-600° C. in air to evaluate the extent of coating of curcuminonto the magnetite nanoparticles. FIG. 4 shows the TGA curve for thesamples. TGA gives quantitative as well as qualitative evidences forcoating of the nanoparticles. It gives an idea about the bondingstrength of the ligand to the nanoparticles, its thermal stability andalso it quantifies the coverage of ligand onto the nanoparticles.Accordingly, thermogravimetric analysis reveals that approximately 45%of curcumin is coated on the nanoparticles. Weight loss corresponding tothe loss of a small amount of adsorbed water is observed in the case ofcoated nanoparticles (CuF) when compared to nearly 20% loss for theuncoated particles (UnF).

The biocompatible, stable curcumin coated ultra-small iron oxidenano-particles synthesized by the process of the instant inventionexhibited reasonable colloidal stability, magnetization as well asfluorescence properties. Curcumin are effectively coated on to themagnetite nanoparticles, in absence of any linker or binder, andsuccessfully retains its hydrogen peroxide scavenging activity.Moreover, the nanoparticles are stable and comparable with that obtainedby using linkers as observed from the relaxivity measurements using NMRtechnique that give values closer to that reported for well-establishedmagnetic resonance imaging (MRI) contrast enhancement agents like citricacid and dextran coated iron oxide nanoparticles.

The present invention relates to a method for magnetic as well asfluorescent imaging, and other biomedical applications comprisingproviding biocompatible, stable, curcumin coated ultra-small iron oxidenanoparticles devoid of any linker or binder as a contrast agent.

The present invention relates to a method for delivering curcumin to asubject in need thereof comprising administering biocompatible, stable,curcumin coated ultra-small iron oxide nanoparticles devoid of anylinker or binder.

The present invention relates to the use of biocompatible, stable,curcumin coated ultra-small iron oxide nanoparticles devoid of anylinker or binder for magnetic as well as fluorescent imaging, and otherbiomedical applications.

The present invention relates to the use of biocompatible, stable,curcumin coated ultra-small iron oxide nanoparticles devoid of anylinker or binder for delivering curcumin to a subject in need thereof.

In sum, the biocompatible, stable curcumin coated ultra-small iron oxidenanoparticles devoid of any linker or binder prepared by the process ofinstant invention provides curcumin coated is suitable for improvedblood residence time, thereby proving to be an efficient candidate fordrug delivery and as contrast agent in MRI, apart from the medicinalproperties of curcumin. Also, the magnetic property of iron oxide coreand fluorescent property of curcumin shell are suitable for use inmagnetic as well as fluorescent imaging, indicating the possiblemultifunctional applications of curcumin coated USPIONs.

The curcumin coated nanoparticles or nanofluids of the invention aresuch that the terminal OH groups of curcumin or its derivatives areretained thereby retaining its medicinal, radical scavenging andfluorescence properties of curcumin.

In another aspect, the invention provides a simple one-pot process forthe synthesis of curcumin or its derivatives coated ultra-smallsuperparamagnetic iron oxide nanoparticles in absence of a linker orbinder comprising adding a solution of curcumin in base to a solution ofmagnetite nanoparticles or nanofluids at low temperature and at pH 9.

The magnetite nanoparticles on which curcumin is coated is preparedusing FeCl₃.6H₂O and FeCl₂.4H₂O as iron precursors, wherein Fe²⁺:Fe³⁺molar ratio is 1:2 and adding the precursors to a base solution,stirring to obtain magnetite nanoparticles and adjusting the pH to 9with a dilute mineral acid.

In an aspect, the biocompatible, stable curcumin coated ultra-small ironoxide nano-particles synthesized by the process of the instant inventionexhibited reasonable colloidal stability, magnetization as well asfluorescence properties. Curcumin are effectively coated on to themagnetite nanoparticles, in absence of any linker or binder, andsuccessfully retains its hydrogen peroxide scavenging activity.Moreover, the nanoparticles are stable and comparable with that obtainedby using linkers.

The curcumin coated iron oxide nanoparticles find application asnanofluid in biomedical applications such as contrast enhancement inMRI, magnetic hyperthermia, drug delivery and cancer treatment.

EXAMPLES

The following examples are given by way of illustration therefore shouldnot be construed to limit the scope of the invention.

Example 1 Synthesis of Curcumin Coated Super Paramagnetic Iron OxideNanoparticles (M1)

To a mixture of 20 ml of iron precursor solution (0.1M FeCl₃.6H₂O and0.05M FeCl₂.4H₂O) dissolved in water was added 100 ml of 10N ammoniumhydroxide under argon atmosphere. The solution was stirred for about 20minutes for complete formation and growth of magnetite particles. Thiswas followed by addition of dilute nitric acid to bring down the pH to˜9. Curcumin solution (0.5 g curcumin dissolved in ammonium hydroxide,pH˜9) was added drop wise to the dispersion. The resultant dispersionwas stirred under argon atmosphere for another 30 minutes and thetemperature of the solution was raised to 80° C. The resulting stablesolution was cooled to room temperature and was dialysed against waterin a cellulose membrane for 3 days to remove excess curcumin followed bydrying in a vacuum oven at 50° C. to obtain the product.

Example 2 Insitu Coated Super Paramagnetic Iron Oxide Nanoparticles (M2)

0.5 g of curcumin was added to 100 ml 10 N ammonium hydroxide solutionand deaerated under argon atmosphere. To this 20 ml iron precursorsolution was added and stirred for 20 minutes for complete formation ofcurcumin coated nanoparticles. The obtained dispersion was dialysed for3 days to remove excess curcumin and then dried in a vacuum oven at 50°C. The sample was labelled as M2. The preliminary characterisation showsthat the phenolic and enolic OH of the curcumin is utilised in bindingwith nanoparticle. Hence further characterisation was done only for M1sample, which is further labelled as CuF.

Example 3 Preparation of Uncoated Super Paramagnetic Iron OxideNanoparticles (Mo)

To a mixture of 20 ml of iron precursor solution (0.1M FeCl₃.6H₂O and0.05M FeCl₂.4H₂O) dissolved in water was added 100 ml of 10N ammoniumhydroxide under argon atmosphere. The solution was stirred for about 20minutes for complete formation and growth of magnetite particles.

Example 4 Characterization

Powder X-Ray Diffraction Studies

Powder X-ray diffraction patterns were recorded on a PAN alytical X'PERTPRO model X-ray diffractometer, in the 2θ range of 10 to 80 degrees.

The uncoated and curcumin coated iron oxide nanoparticles werecharacterized using powder X-ray diffraction (XRD).

XRD patterns of standard JCPDS data (19-0629) of Fe₃O₄ (a), M0 (uncoatedsample, UnF), M1 (post synthesis functionalised magnetite nanoparticles,CuF) and M2 (insitu coated nanoparticles)

The crystallographic information of the synthesized nanoparticles wasobtained from X-ray diffraction studies. The X-ray diffraction studiesrevealed that the iron oxide phase formed was biocompatible magnetiteand the crystallite size obtained was around 5 nm. Uncoated magnetitenanoparticles were also synthesized by the same procedure for comparisonthe XRD pattern of the as synthesized nanoparticles is shown in FIG. 1.

The uncoated (M0, UnF) and curcumin coated (M2, CuF) iron oxidenanoparticles were characterized using powder X-ray diffraction (XRD).The XRD patterns of the uncoated (UnF) and curcumin coated (CuF) samplesmatched well with the standard pattern of magnetite (JCPDS #19-0629).The average crystallite size was calculated using the Scherrer formula,D=0.91λ/β cos θ, where λ is the wavelength of X-rays (Cu Kα=1.542 A°), βis the full width at half maximum (FWHM) after correcting for theinstrumental contribution, and θ is the Bragg angle. The averagecrystallite sizes of uncoated and coated samples were obtained as 7 nmand 4 nm, respectively.

TEM Image

TEM analysis was performed on a FEI, TECNAI G2 TF30 instrument. Sampleswere prepared by placing a drop of dilute dispersion on carbon coated200 mesh copper grid and imaged at an accelerating voltage of 300 kV.

The TEM image of the curcumin coated particles (M2, CuF) show isolatedparticles (FIG. 2) with average size of ˜3 nm which was in closeagreement with average crystallite size calculated from XRD. On theother hand, the TEM image of the uncoated sample (M0, UnF) showed highlyagglomerated clusters. This indicated that curcumin was efficientlychemisorbed on a nanoparticle and was sterically separating theindividual particles in the system.

IR Analysis

IR studies were carried out on a Perkin Elmer Spectrum-One FTIRspectrometer in the frequency range of 400-4000 cm⁻¹ by properly mixingthe sample with spectroscopic grade KBr.

The presence of curcumin on the surface of the magnetite nanoparticleswas qualitatively monitored using IR spectroscopy. The broad band at 590cm⁻¹ was observed due to the Fe—O—Fe stretching vibration of magnetite.A comparison of the spectra of the coated sample and curcumin shows thatmost of the IR bands of curcumin were present in the spectra of thecoated particles. However, curcumin coated iron oxide nanoparticles (M2,CuF) showed significant changes in the position of various bands ofcurcumin. The intensity of the sharp band at 3510 cm⁻¹, corresponding to2-phenolic OH and 1-enolic OH was decreased after coating on thenanoparticles. The reduced intensity of the band indicated that some ofthe OH groups of curcumin are free in the functionalized sample. The pHadopted for the reaction was about 9, at which only the enolic OH willbe ionized, whereas at a pH above 9 all the three OH will be ionized.The C═O stretching band at 1628 cm⁻¹ of curcumin was shifted to 1621cm⁻¹ indicating that a chemical bond was formed between nanoparticlesurface and the ligand through the enolic OH. The bands corresponding toC═C stretching and —C—C═O in-plane bending also shifted slightly,whereas there was no considerable shift for the in-plane bending of C—CHand C═CH of the aromatic ring (FIG. 3).

The bands relating to the aromatic ring were not at all affected aftercoating which suggested that the OH group on the aromatic ring remainedintact without taking part in the coating.

Thermogravimetric Analysis

Thermograms of the synthesized samples were recorded on a Perkin ElmerTGA7 analyzer in air.

Thermogravimetric analysis (FIG. 4) of the as-synthesized sample wascarried out from room temperature to 600° C. to know the extent ofcoating. The thermogram shows that approximately 45% curcumin was coatedon the nanoparticles. Weight loss corresponding to the loss of a smallamount of adsorbed water was observed in the case of coatednanoparticles when compared to nearly 20% loss for the uncoatedparticles.

Room Temperature Magnetic Measurements

Magnetic measurements were done on a Quantum Design MPMS 7T SQUIDVSM.Zero field cooled (ZFC) and field cooled (FC) magnetization measurementswere done at 50 Oe and room temperature magnetic measurements were donefrom −3 T to +3 T.

Room temperature magnetic measurements (FIG. 5) of the curcumin coatedand uncoated samples show that the magnetization of both uncoated andcoated samples did not get saturated even at a magnetic field of 3 T.Also, no hysteresis loops were observed for both samples (zerocoercivity), indicating that both samples are super paramagnetic. Themagnetization at 3 T for M0 (UnF) was 30 emu/g whereas that of thecoated sample (M2, CuF) was 11 emu/g. The decrease in the magnetizationof coated nanoparticles compared to that of the uncoated sample can beattributed to the presence of the non-magnetic surface curcumin layerover the nanoparticles as well as the smaller particle size. Further,the effectiveness of coating of curcumin on the nanoparticles wasstudied by temperature dependent magnetic measurements. Theinter-particle magnetic interactions (both dipolar as well as exchange)was suppressed if the coating was effective.

Direct comparison of the nature of the zero field cooled (ZFC) and fieldcooled (FC) magnetization curves provided information on theinter-particle interactions, and particle size distribution. The ZFC andFC magnetization curves were measured in a field of 50 Oe (FIG. 6). Inthe ZFC measurement, when the particles are cooled to the lowesttemperature, the magnetic moments of individual particles are randomlyoriented and the magnetization tends to be zero, and once when a fieldis applied while heating back to room temperature, the moments tend toalign in the direction of the magnetic field up to a specifictemperature known as the super paramagnetic blocking temperature. Thetemperature was proportional to the volume of the particles through therelation, KV≈25k_(B)TB where V is the volume of a particle and TB is theblocking temperature. K is the total magnetic anisotropy from variouscontributions, and k_(b) is the Boltzmann constant. Above thistemperature, the thermal energy was sufficient enough to overcome theanisotropic energy and hence the magnetization decreased. Narrowparticle size distribution resulted in a narrower peak in the ZFCmagnetization curve and a broad peak was expected when the sizedistribution was wide. The narrow ZFC curve of coated sample, comparedto the broad curve for uncoated iron oxide particles, explained monodispersity of the system, which was clearly reflected in the TEM image.The FC curve remains almost constant below the blocking temperature forthe uncoated sample whereas the magnetization decreases continuouslyafter coating. This is an indication for the reduced inter-particleinteractions after coating. Similarly, the lower blocking temperature ofthe coated sample was also an indication for the reduced anisotropycontribution from inter-particle interactions. Thus, the magneticstudies indicate that curcumin was effectively coated on the iron oxidenanoparticles.

Hydrogen Peroxide Scavenging Assay of Curcumin Coated Iron OxideNanoparticles

1 ml solution of 40 mM H₂O₂ in phosphate buffer (pH=7.1) was added to 4ml (15 ppm) dispersion of the coated nanoparticles in phosphate buffer(pH=7.1). The mixture was incubated for 15 minutes and the absorbancewas measured at 230 nm which is the lmax of hydrogen peroxide. Thepercentage of H2O₂ scavenging by the sample was calculated using theequation,H₂O₂ scavenging effect (%)=(1−A _(s) /A _(c))×100where A_(c) is the absorbance of the control and A_(s) is the absorbanceof the sample.

The scavenging activity of the coated sample was obtained as ˜62%compared to the 81% scavenging activity of 30 ppm curcumin solution.This showed that curcumin retained its radical scavenging property evenafter coating on the SPIONs, confirming that the anti-oxidant activityof curcumin was retained after coating. The phenolic hydroxyl group isknown to be responsible for the strong anti-oxidant property ofcurcumin, and the present study confirms that the phenolic hydroxylgroup is remaining free on the curcumin coated on the nanoparticles,supporting the conclusions from other studies. The XRD pattern of thesample after H₂O₂ scavenging activity assay was identical to that of theuntreated sample, indicating that the hydrogen peroxide did not interactwith the magnetite core. Similarly, the IR and UV-Visible spectra ofhydrogen peroxide treated and untreated samples were also found to besimilar. The hydrogen peroxide scavenging assay was further performed onthe treated sample, which gave a scavenging activity closer to thatobtained initially. The XRD pattern, IR and UV-visible spectra of thesample again gave a similar result to that of the first one. Theseresults confirmed that the curcumin coated nanoparticles are highlystable and could be reused for peroxide scavenging as well as for otherapplications, since there was no structural damage observed after theperoxide treatment.

UV-Visible and Fluorescence Spectra of the Curcumin Coated NanoparticlesDispersed in Ethanol are Compared with that of Pure Curcumin.

Curcumin shows a peak at 425 nm in UV-visible spectra corresponding tothe π-π* transition. The curcumin encapsulated samples shows abathochromic shift of about 4 nm as shown in (FIG. 6A). The shift in thepeak position is attributed to the changes in the environment ofcurcumin, as evidenced from the IR spectra. The emission property offluorescent CUR molecule is known to depend on the polarity of itsenvironment. Fluorescence spectra of the coated sample (FIG. 6B) show asmall red shift which results from the polar environment aroundcurcumin. This further confirms that the curcumin molecule is coated onthe SPIONs

Magnetic Resonance Imaging (MIR)

Magnetic resonance imaging demands the use of contrast agents forenhancing the quality of images. The role of the contrast agents is toefficiently vary the relaxivity of water protons. The relaxation ofprotons occurs by two pathways namely the longitudinal (T1 relaxation)and transverse (T2 relaxation). Depending on these relaxation processes,the contrast agents are classified as T1 and T2 contrast agents.Gadolinium complexes act as T1 contrast agents whereas iron oxidenanoparticle efficiently alters the T2 values of water protonssurrounding the particle. The magnetic nanoparticles in a tissueenvironment when subjected to a magnetic field produce a heterogenousfield gradient allowing the water protons to diffuse. The dipolarcoupling between the magnetic moments of water protons and that of theparticles lead to spin dephasing and T2 relaxation, resulting in reducedsignal intensity, and hence termed as negative contrast enhancement.

Iron oxide nanoparticles with core diameter less than 10 nm (USPION)produces positive contrast enhancement in T1 weighted images. Iron oxidenanoparticles with effective surface coating was observed to be apromising candidate as contrast agents in MRI as they do not need atargeting probe to image tumors. T1 and T2 measurements were carried outat 400 MHz for three different concentrations of the nanoparticlesdispersed in DMSO. The corresponding relaxation rate enhancements of thesuspension, R1 and R2, which represent per millimolar concentration ofFe ions present was calculated by determining the number of magnetic Feions per particle. The calculation is based on the consideration thateach unit cell has eight magnetic ferric ions. The relaxivities of thesynthesized SPIONs were calculated from the T1 and T2 values, byaccounting for the number of magnetic iron ions present per particle.The normalized values were obtained as 30407 s⁻¹mM⁻¹ for transverserelaxivity and 126 s⁻¹mM⁻¹ for longitudinal relaxivity (FIG. 7). Since a3 nm Fe3O4 contains ˜192 Fe3+ ions contributing to the magnetic moment,longitudinal R1 and transverse R2 relaxivities were calculated as 0.66s⁻¹mM⁻¹ and 158 s⁻¹mM⁻¹ respectively. The R2/R1 value was calculated as240 at 400 MHz which was much larger than the minimum threshold value(=2) required for effective negative contrast. This indicates that thecurcumin coated USPION is a suitable candidate for use in MRI as acontrast agent. The R2/R1 ratio greatly depends on the applied magneticfield. However, not much variation was observed for citrate coatednanoparticles when measured at the normal MRI frequency range of 60 MHzand at 300 MHz which is close to the frequency used for the presentmeasurement (FIG. 7). The ratio gives information on how much nuclearlongitudinal magnetization is available after each subsequentacquisition for averaging the signal, and the ratio depends on themagnetic moment of the single particle. The relatively larger value ofthe relaxivity ratio for the curcumin coated sample indicates that it isa better contrast agent for MRI with the additional benefits of themedicinal and fluorescence properties of curcumin.

ADVANTAGES OF THE INVENTION

-   -   1. Simple one pot process    -   2. Biocompatible iron oxide nanoparticles synthesized, as it        does not require any harmful precursors    -   3. It may be used for contrast enhancement in MRI, magnetic        hyperthermia, drug delivery, cancer treatment and other related        applications    -   4. It may be used equally for in-vivo as well as in-vitro        applications

We claim:
 1. Curcumin coated magnetite nanoparticles comprisingbiocompatible, stable curcumin coated ultra-small super paramagneticiron oxide nanoparticles devoid of any linker or binder, the curcumincoated magnetite nanoparticles having a particle size of about 3 nm,wherein the curcumin coated magnetite nanoparticle retains medicinal,radical scavenging and fluorescence properties of curcumin, forbiomedical applications, wherein curcumin is directly coated onultra-small super paramagnetic iron oxide nanoparticles.
 2. A method formagnetic resonance or fluorescent imaging, the method comprisingadministering curcumin coated magnetite nanoparticles of claim 1 to asubject, and performing an imaging step.
 3. A method for deliveringcurcumin to a subject in need thereof comprising administering curcumincoated magnetite nanoparticles, of claim
 1. 4. The curcumin coatedmagnetite nanoparticles of claim 1, which are biocompatible, stablecurcumin coated ultra-small iron oxide nanoparticles devoid of anylinker or binder for use as a contrast agent for magnetic resonancefluorescent imaging.
 5. The curcumin coated magnetite nanoparticles ofclaim 1, which are biocompatible, stable, curcumin coated ultra-smalliron oxide nanoparticles devoid of any linker or binder for use indelivering curcumin to a subject in need thereof.